Yeast Artificial Chromosome (YAC) Clones

Ramaiah Nagaraja, National Institute on Aging, Baltimore, Maryland, USA
David Schlessinger, National Institute on Aging, Baltimore, Maryland, USA

Published online: September 2005

DOI: 10.1038/npg.els.0005347

Artificial chromosome vector systems have facilitated mapping and sequencing of complex genomes at an increasingly rapid pace. They include cloned DNA fragments ranging from 50 kb to more than 1 million base pairs, as well as sequences that render them capable of growth in yeast or bacteria. Traditional bacterial cloning systems have remained important for the study of relatively short clones, but for the cloning of very large DNA segments artificial chromosomes have completely replaced earlier bacterial systems, including lambda phage-based cosmids.

Keywords: YACs; BACs; PACs; artificial chromosomes; cloning vectors

Figure 1. Generalized features of a typical YAC cloning vector and cloning steps. The YAC vector has yeast selectable auxotrophic markers for tryptophan (TRP1) and uracil (URA3) biosysnthesis. The centromere (CEN4) sequence is derived from yeast centromere 4. Cleavage with BamHI and EcoRI releases two arms of the YAC vector terminating in Tetrahymena pyriformis telomere (TEL) sequences associated with one of the selectable markers and disrupts the SUP4 gene that suppresses the ADE4 mutation. The stuffer fragment released by digestion with BamHI between the telomeres is removed during purification of the arms. The genomic DNA is digested with EcoRI in the presence of EcoRI methylase to prevent excessive digestion, fractionated by pulsed field gel electrophoresis (or zonal sedimentation through a sucrose gradient) and ligated to the arms and transformed into Saccharomyces cerevisiae strain AB1380. Mutation in the ADE4 gene in the host strain yields transformed colonies that are CEN4 in the absence of the SUP4 gene on a selection medium lacking uracil and tryptophan. Such colonies are further purified and the presence of insert verified.
Figure 2. Principles of transformation-associated recombination (TAR) cloning. A BAC-based vector containing yeast centromere (CEN) and selectable marker (HIS3) sequences is cleaved with a restriction enzyme at a unique site placed between the ‘hooks’ (DNA sequences of at least 200 bp at the edges of the region to be cloned). The digested vector is transformed into yeast along with high molecular weight genomic DNA. The ‘hooks’ pair with the corresponding sequences in genomic DNA, and recombination produces the TAR clone. The cells are plated and recombinants that grow depend on the presence of replication origins in the cloned DNA, thus ensuring that the DNA between the ‘hooks’ is rescued. Note that this vector system rescues genomic DNA in circular form; unlike YAC construction it does not require the presence of telomere sequences, and discourages cocloning events; in fact, no cocloned TAR-derived YAC has been observed.
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 References
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    book Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, a Laboratory Manual, 2nd edn, vol. 1, pp. 2.2–3.58. Plainview, NY: Cold Spring Harbor Laboratory Press.
 Web Links
    ePath Vector Information. For a description of PAC/BAC vectors, thier sequences, and library resources. http://www.chori.org/bacpac/vectors.htm

Related Sub-Topics:

Replication and Recombination | Techniques and Tools in Clinical Genetics | Techniques and Tools in Molecular Biology
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Article Title: Yeast Artificial Chromosome (YAC) Clones
 
How to Cite close
Nagaraja, Ramaiah, and Schlessinger, David(Sep 2005) Yeast Artificial Chromosome (YAC) Clones. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005347]